The long-term objective is to characterize mechanisms by which a host-derived protein called PINCH promotes neuronal fitness in response HIV infection of the CNS with the overall goal to understand PINCH's role in neurons exposed to viral and inflammatory factors. Synaptodendrtic injury correlates closely with the presence and severity of cognitive impairment in HIV patients. Since synaptodendritic damage in HIV infection of the brain is partially reversible and can occur in the absence of significant neuronal loss, information gained from these studies will enhance our ability to improve therapies to treat HIV infection of the brain and alleviate neurological complications observed in many HIV patients. In the brains of HIV infected individuals, PINCH is expressed exclusively by neurons showing signs of synaptodendritic damage. PINCH is an adapter protein that mediates bidirectional signal transduction between the extracellular matrix and intracellular networking pathways via interactions with integrin linked kinase (ILK) and Nck2. Thus, our overall hypothesis is that in response to disruptions in neuronal signaling caused by HIV infection of the CNS, PINCH protein is stabilized by sumoylation to promote proper interactions with ILK and Nck2. We propose two AIMS that investigate in vitro I) mechanisms by which PINCH expression is regulated, and II) the biological consequences of PINCH expression on neurons exposed to viral protein and host inflammatory factors produced during HIV infection of the CNS. Primary neurons treated with TNF-a or Tat to mimic some aspects of HIV infection of the brain will be assessed for PINCH expression via qRT-PCR, and Northern and Western analyses, and by siRNA knockdown. Sumoylation of PINCH will be assessed by reciprocal IP and Western analyses after exposure to TNFa and/or Tat. We further hypothesize that disruptions in PINCH's normal communication with ILK and Nck2 may diminish neuronal recovery. Neurons exposed to TNFa and/or Tat will be assessed for ILK and Nck2 expression and functional interactions with PINCH. To address biological consequences of PINCH in the context of HIV infection of the CNS, site-directed mutagenesis, PINCH protein accumulation and MALDI-TOF analyses will be used. Expanded studies of HIV patients' brains with MCMD, HIVE, dementia and importantly HAND in the absence of HIVE as well as HIV patients who died without CNS complications will be analyzed via laser capture microdissection of neurons and MALDI-TOF analyses will also be conducted.